Physiology is no certain method, but a kind of question: We use a wide
spectrum of different methods - always aiming to answer the question "How
does it work?" with respect to the investigated molecules, cells, organs
or the whole body.

This question cannot be fully answered using classic, e.g.
electrophysiological, methods alone without knowing about the molecular
structures. On the other hand, molecular biology and biochemistry results
usually do not offer conclusions regarding the function of the whole cell
or the intact body. In our current projects we therefore combine
functional (= mostly electrophysiological) and structural (biochemical,
molecular biological, laser-scanning microscopical, and electron
microscopical) techniques.

In order to understand the pathophysiology of a disease it is
necessary to know the mechanisms of normal function. For most topics we
study the basic mechanisms and - using the same methods - the impaired
function in diseased states. Experiments are performed on isolated
epithelia and cell cultures. Within this spectrum of approaches we
collaborate for many years with the Dept. of Gastroenertology (Head: Prof.
Britta Siegmund).

Our
main research topic is the tight junction:

The intestinal barrier critically depends on the function of the
tight junctions. Using translational approaches, we study functions,
regulation, and structure of the tight junction proteins in health and disease. Methods are molecular biological,
microbiological, (ultra-)microscopical, biophysical, and electrophysiological.

Tight junction proteins comprise three (even four if one includes JAM) families. All are transmembrane proteins
having one important feature in common: They are arranged in such a way that they interact with other TJ proteins of the
neighboring cell and by this are able to seal the cleft between these cells against unlimited passage of solutes and
water.

Two of these families possess four transmembrane
domains (tetraspan proteins).

Most claudins and TAMPs act as barrier formers within the TJ.
In contrast, some claudins form paracellular channels
through the TJ which are selective for cations (claudin-2, claudin-10b, claudin-15), anions (claudin-10a, claudin-17) or water
(claudin-2).

All tetraspan TJ proteins comprise intracellular N- and C-terminals, one small intracellular loop, and two extracellular loops (ECL1
and -2). ECL1 is thought to determine the paracellular barrier and/or channel function. ECL2 may act as mechanical
contact between opposing tight junction proteins. The exact molecular
structure of some proteins is partly to almost fully resolved.

Another two families of TJ proteins contain one transmembrane domain:

Angulin family. They comprise three TJ
proteins, LSR, ILDR1, and ILDR2 and are mainly localized at the tricellular tight junction.

Localization: Typical for
epithelia with almost impermable tight junctions ("tight epithelia") like distal
colon and distal kidney tubuleFunction: Paracellular barrier against
transepithelial diffusionClinical Impact: Claudin-1 is reduced in
hereditary mammary cancer. A defect of claudin-1 is present in neonatal
sclerotic cholangitis with ichthyosis. Expression of claudin-1 and of claudin-4
was increased in colorectal cancer and pancreatic and ovarian cancers. In human
epidermis claudin-1 is the essential paracellular barrier former

Claudin-3Localization: Typical for tight
epithelia
Function: We have characterized
claudin-3 to be a general barrier former as it reduces permeability for ions
without charge preference and uncharged solutes.Clinical Impact: Claudin-3 and -4 are
receptors for the enterotoxin of Clostridium perfringens.

Claudin-5Localization: Typical for endotheliaFunction: We were able to show that
claudin-5 belongs to the barrier-forming claudins and that it is expressed also
in some epithelia. Clinical Impact: Claudin-5 is deleted in
patients suffering from velo-cardio-facial syndrome (DiGeorge syndrome).
Claudin-5-deficient mice exhibit a barrier defect of the blood-brain barrier.

Localization: Claudin-10 exists
in six splice variants. Two variants are of major importance, 10a, localized in
kidney proximal tubule TJs, and variant 10b, localized in many epithelia including kidney
Function: Claudin-10a is anion selective. Claudin-10b forms a
cation selective channel, which is, in contrast to claudin-2, not permeable
to water.

TAMP
familyGeneral:TAMP stands for Tight
junction-Associated Marvel Proteins. The TAMP family
includes occludin, tricellulin, and marvelD3, which share a transmembrane domain
motif called MARVEL (Myelin and lymphocyte And Related
protein for VEsicle trafficking and membrane Link).

OccludinLocalization: All epithelia
Function: The function of occludin is still poorly understood. In a
collaboration, the lab of Shoichiro Tsukita and our group have shown that in
occludin-KO mice the tight juncion barrier is unaltered. This means that
occludin either has no intrinsic barrier properties or can be replaced by other
components of the tight junction.

In occludin-knockout mice the
glandular structure of the stomach exhibited a complete loss of parietal cells
and mucus cell hyperplasia, as a result of which acid secretion was virtually
abolished. A dramatic change in gastric morphology and secretory function
indicates that occludin is involved in gastric epithelial differentiation.

Little is known about the regulatory mechanisms of occludin that influence
occludin gene expression. We aimed to identify the sequences essential in cis
for genomic regulation of tight junction formation and to investigate their
functional role in cytokine-dependent tight junction regulation.
Using genome walking cloning of occludin-specific human genomic DNA sequences, a
1853 bp DNA fragment containing the transcription start point of occludin cDNA
sequences was amplified and sequenced. The proinflammatory cytokines, TNFa
and interferon g diminished occludin promoter
activity alone and even synergistically, suggesting a genomic regulation of
alterations of the paracellular barrier. Both cytokines downregulate the
expression of occludin, paralleling the barrier disturbance detected
electrophysiologically. This could be an important mechanism in gastrointestinal
diseases accompanied by barrier defects, for example inflammatory bowel
diseases.

Tricellulin (= marvelD2)Localization:Tricellular tight junction (tTJ), i.e. the site
where three epithelial or endothelial cells meet.Function: Tricellulin was discovered by Shoichiro Tsukita who has died in Dec. 2005,
a few days before his landmark paper appeared: Ikenouchi et al., 2005, J. Cell Biol. 171(6): 939-945 [PubMed]
[PDF].In cell
cultures, lack of tricellulin prevents the development of the epithelial
barrier. We showed that tricellulin tightens the tricellular junction against
macromolecules. We propose that, at impaired tricellulin expression, the tTJ
becomes a major site for the passage of macromolecules.

JAM proteins are localized just "below" the TJ strands (meaning more to basal cell side) and provide
mechanical adhesion between
lateral membranes of neighboring cells. JAM molecules have no direct barrier function by itself, but if JAM cell-cell
contacts are impaired the lateral cell membranes lose contact. Necessarily, adjacent TJ proteins also lose contact to
each other and the paracellular barrier opens.

JAMs form cis- and trans-interactions with other JAMs. All JAMs contain PDZ motifs and bind to numerous intracellular
parters.

Scaffold proteins provide an intracellular connection between most claudins and the TAMPs with the actin cytoskeleton. Best-known are ZO-1 and ZO-2 (Zonula Occludens-1
and -2). The name "Zonula occludens" suggests they are tight junction proteins, but in a strict sense they are not. They are located intracellularly and connected via PDZ domains with the claudins,
TAMPs, and JAMs.

The first crystal structure of a claudin was published in 2014 by Suzuki et al. for
claudin-15 [PubMed].

This was a major breakthrough, which then also allowed for homology studies
on other members of the claudin family.We have performed studies on the molecular structure of the following
claudins:
- Claudin-3 (Rossa, Plöger et al. 2014; Milatz et al. 2015)
- Claudin-5 (Rossa, Protze et al. 2014; Rossa, Plöger et al. 2014)
- Claudin-17 (Conrad et al. 2016)

The crystal structure of tricellulin and its molecular
architecture within the tricellular tight junction is not yet resolved.
In cooperation with the MDC, we have analyzed part of the protein, the C-terminal domain.

This passage is cited from Krug et al. 2014: "Epithelia form barriers against unlimited passage
of solutes and water, but also regulate and allow distinct permeation across that barrier. On the one hand, such
permeation sites are located within the cell membranes, forming a transcellular pathway via channels, carriers, and
transporting ATPases. On the other hand, the paracellular pathway between the cells is sealed against uncontrolled
passage by the TJ.

However, long before claudins and TAMPs were identified as constituents of the TJ it was demonstrated
that the paracellular pathway of some, but not all, epithelia is permeable to small ions [Frömter
& Diamond 1972]. This in mind, the concept of “leaky” and “tight epithelia” was born [Diamond
1974]: in leaky epithelia the paracellular pathway is more ion-conductive than the transcellular one. In intestine
and nephron, leaky epithelia are typically found in proximal segments. Tight epithelia behave the other way around and
in intestine and nephron they are present in distal segments.

While many TJ proteins indeed have barrier-forming properties, there are also several claudins forming
charge- and/or size-selective paracellular channels. These channels are not crossing membranes as transmembranal
channels do, but are orientated parallel to the lateral membranes allowing permeation through theTJ. They are formed by
the extracellular loops of TJ proteins interacting with extracellular loops of TJ proteins located in the opposing cell
membrane.

Often there are uncertainties whether the conductive claudins should be named channels or pores. Simply
said, both is correct: the pore is one part of a channel.
A channel is the entity of a permeation site comprising
(i) a pore,
(ii) a narrow site that restricts access by size and shape (size selectivity),
(iii) a site that favors passage by charge or charge density (charge selectivity), and
(iv) a feature providing time-variant permeability changes (gating).

By definition, “selective for x” means that the permeability for x is higher than that for other
substances or groups of substances. All channel-forming claudins exhibit at least one of the three types of selectivity:
for cations (claudin-2, claudin-10b,
claudin-15), for anions (claudin-10a,
claudin-17) or for water (claudin-2).

Charge selectivity cannot be determined from transepithelial resistance (TER)
but from dilution potential measurements. Here, charge selectivitiy is read out from the resulting ratio PNa/PCl.
PNa>PCl indicates cation selectivity and PNa<PCl indicates anion selectivity
[Günzel et al. 2010; Yu et al. 2009]. Ratio changes together with the calculated absolute permeabilities give
information about the preference. Higher selectivity, as e.g. exclusively for sodium only can be found in some membrane
channels like the epithelial sodium channel ENaC, but yet not for any TJ protein.
Thus, TJ protein channels formers and also barrier formers exhibit substrate-specific transmissive properties.

Therefore, the term “permeability” is incomplete without relying to the analyzed substance(s) for which
the TJ protein is transmissive."

For many years there had been a dispute regarding the contribution and even existence of paracellular water transport.
It was in 2010 when this dispute ended after we discovered that it is claudin-2 that forms a water channel
(Rosenthal et al. 2010). We showed that the claudin-2-based pore is permable to cations (Amasheh et al. 2002)
as well as to water (Rosenthal et
al. 2017). However, the ion permeability of other claudins is not necessarily coupled to water permeability: the cation channel
claudin-10b and the anion channel claudin-17 proved to be not water permeable.

The tricellular tight junction (tTJ) is localized at contacts of three epithelial or
endothelial cells. Here, the elements of adjacent bTJ strands join and extend in basal direction. Importantly, the
tTJ forms a vertical central tube which is considered to be a structural weak point of the whole TJ network.
Proteins found predominatly at the tTJ are tricellulin and the
angulins.

Tricellulin plays a critical role for barrier formation against macromolecule passage.
This means that at low abundance of tricellulin the passage of medium-sized and large molecules
will facilitated in this region (Krug et al. 2009). Of course, if any pathway opens for large molecules also small
molecules and ions would pass, and this becomes numerically relevant in tissues low in claudin ion channels, i.e.
"tight" epithelia (Krug 2017, Ann. N.Y. Acad. Sci.). The opening of the tTJ may occur in an unwanted or an intended
manner:

Unwanted opening of the tTJ: In first studies with human colon biopsies we have shown that tricellulin is
downregulated in the inflammatory bowel disease (IBD) ulcerative colitis and the tTJ is opened (Krug et al. 2017,
Mucosal Immunol.). We hypothesize that this causes luminal pathogens to pass which then
supports the inflammatory process (Krug et al. 2014). A role of other proteins, which are located at the tTJ, e.g.
angulins (LSR, ILDR1, ILDR2), as well as for occludin might be assumed (Martini et al. 2017).

Intended opening of the tTJ: In a novel approach Masuo Kondoh and we developed a
paracellular drug absorption enhancer acting at the tTJ, named angubindin-1. Its binding led to removal of angulin-1 and
tricellulin from the tTJ which enhanced the permeation of macromolecular solutes (Krug et al. 2017, J. Contr. Release).

If the ion permeability is critically increased under pathological conditions
a leak flux diarrhea occurs.
This type of diarrhea is caused by massive fluxes of solutes and water from the
blood into the gut lumen.

Regarding immunological mechanisms, an intact epithelial barrier keeps
luminal bacteria, toxins, and antigens away from the subepithelial tissues. It
is discussed, whether an impaired intestinal barrier allows for increased uptake
of bacteria, toxins, and antigens which then will support the inflammation
process.

Cytokines like tumor necrosis factor alpha (TNFa),
interleukins and interferons act as mediators of inflammation. In inflammatory
bowel diseases (and in HIV infection) their local concentrations increase. We
study the action of cytokines on transport and barrier function of human
intestine and cell cultures originating from human colon (HT-29/B6).

The lumen of the small and especially the large intestine is populated by an
unimaginable

number of bacteria. Most are good-natured and help digesting food. Normally, they remain in the gut lumen and do not
pass the intestinal wall, except the epithelial barrier is injured. However, some other bacteria are able to produce
their own pathway across the gut wall. After wall passage they may act as pathogens, maintaining, enhancing or even
initializing intestinal inflammation. The mechanisms by which pathogens can translocate are mainly paracellular,
including (i) focal leaks, (ii) epithelial apoptosis, and (iii) opening the tight
junction pathway, espectially at the tricellular tight junction.

In celiac disease, a T-cell-mediated response to gluten occurs in genetically predisposed individuals. Gluten is found
in grains like wheat, barley, and rye. Importantly, gluten proteins contain peptide sequences which can elicit T-cell
responses in the small intestine. This results in a malabsorptive enteropathy characterized by villus atrophy and
crypt hyperplasia. The barrier defect includes altered tight junction proteins.